18th Annual InternationalConferenceof the IEEE Engineeringin Medicine and Biology Society, Amsterdam 1996
6.2.8: Physiological Modelling - Neuromuscular General
Modification of Pattern of Excitatory Junction Currents in
the Guinea Pig Vas Deferens by the Gap Junction Uncoupler Heptanol
Manchanda, R. and Venkateswarlu, K., School of Biomedical Engineering,
Indian Institute of Technology, Powai, Mumbai - 400 076, India.
Abstract - Extracellularly recorded excitatory junction
currents (EJCs) produced by neurotransmission to the
smooth muscle of the vas deferens have a varied phasic
composition. They can be positive going (p-EJC), negative
going (n-EJC) or biphasic (b-EJC) from one event to the
next. The phasicity is believed to depend upon the syncytial
properties of the smooth muscle cells. We have investigated
the effects of l-heptanol, an intercellular uncoupling agent,
on the phasicity of EJCs. In control Krebs solution EJCs
with a positive phase (p- and b-EJCs) occurred as a considerable fraction of total EJCs recorded. Following the application of 2 mM heptanol for 2-3min., the Occurrence of pand b-FJCs decreased markedly while that of n-FJCs increased. The change was reversible and repeatable. Heptanol did not, however, affect the prejunctional nerve action
potential. These bioelectric results can be explained in terms
of the specific uncoupling of smooth muscle cells by heptanol.
I. INTRODUCTION
Individualsmooth muscle cells are coupled to one another to
formathreedimensionalsyncytium[ 1l.Thepattemofelecuical
activityduring stimulationevoked neurotransmissionin smooth
muscle,as recorded with a focal extracellularelectrode,depends
upon the pattern of ongoing neurotransmitter action within and
around the circumference of the electrode [2], [3]. Negative
going excitatoryjunction currents (n-FJCs)arerecorded if excitatory transmitter action occurs at neuromuscular junctions
(NMJs) within the electrode. If however activity at NMJs just
extemal to the rim of the electrode predominates, then positive
EJCs (p-HCs) are produced [2]. Finally, when NMJs both
within and outside the electrode are simultaneously active, a
biphasic EJC (b-ETC) can result. The positive phases in p- and
b- ElCs are believed to arise due to local current flow between
smooth muscle cells, from those outside the electrode into cells
underneath the electrode, through intercellular electrical pathways. In this paper we report that heptanol, which blocks intercellular electrical communication [4], [5], changes the phasic
composition of EJCs in a way that is consistent with its uncoupling action.
11. METHODS
Vasa deferentia were dissected out from adult male Hartley
guinea pigs along with the hypogastric nerve. Recordings were
made in vitro in a perspex organ bath in which the tissue was
mounted and superfused with Krebs solution at 2-3 ml/min.
Solutionsof 1-heptanol were made up by vigorous shaking with
Krebs at the time of experiment. The nerve was stimulated via
0-7803-381 1-1/97/$10.00 QIEEE
AgJAgC1 ring electrodes, using rectangular voltage pulses
(amplitude 2-10 V, width 0.05 - 0.5 ms at 0.7 Hz). Focal extracellularmrdingsof nerveactionpotentialsandFJCsevoked
by stimulation were made using glass microelectrodes with tip
diameters of 50-100 pm and filled with normal Krebs, using
conventional methods [23. Signalswere led to an a.c. amplifier,
displayed on a storage oscilloscope and stored on tape. They
were collected on an IBM PC-AT compatible using an A/D
conversion card and analysed with the help of custom made
software [61.
1II.RESULTS
Fig. 1 provides examples of the pattem of EJCs recorded
before and during the applicationof heptanol in one mal. prior
to the addition of heptanol all three kinds of EJCs (n-, p- and b) were recorded (Fig. 1Aa). The Occurrence of each kind in a
seriesofeventswas random. Their proportions are shown in the
columnofFig. lB.Onaveraging35 suchEJCsabiphasicrecord
was obtained (Fig. 1Ca). Application of heptanol (2.0 mM)
caused a rapid, profound change in the proportion of each kind
of ETC. The occurrence of p-EJCs was gradually suppressed
and finally abolished, while n-EJCs became increasingly frequent (Fig lA, B). Consequently,the averaged H C showed a
correspondingshift in phasicity from being biphasic towards increasing negativity (Fig. IC). Theaverage peak positiveamplitude was reduced from 36k3 pV (control,n=80) to 0 pV (3 min.
heptanol, n=30) and average peak negative amplitude was increased from -33k2 pV to -83k4 pV.The changes werereversible (Fig. Id) and repeatable (3 trials).
In contrast, heptanol was found not to affect any of the
principal features@e& amplitude,timecourse,duration)of the
axonalaction potentialprecedingthe EJC, when recordedeither
with the FJC (Fig. IC) or separately (Fig. 2).
IV. DISCUSSION
While much workhasbeenreportedontheeffectsofheptanol
at the single-celllevel [5], [7], and on smooth muscle contractions [4], its effectson the electrophysiologyof neurotransmission have not previously been explored. The change in phasic
composition of the EJC in the presence of heptanol observed
here can be explained on the basis of its electrical uncoupling
action. During the n-EJC the recording electrode becomes
negativeowing to the net removal of positive ionic charge from
the extracellular fluid into smooth muscle cells because of
excitatory transmitter action at NMJs underneath the electrode.
However, if NMJs outside the electrodeare active, net positive
charge is injected into cells surroundingthe electrodeand may
spread through intercellularjunctions into the volume of tissue
1769
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18th Annual International Conference of the IEEE Engineering in Medicine'and Biology Society, Amsterdam 1996
6.2.8: Physiological Modelling - Neuromuscular General
c
A
b
H e w l
b-
48
40
(1.5 min.)
d
Wash out
(6.5 min.)
.....
n.a.p
Fig.I Consecutiveindividual samples (A)and averages (C)of UCs before (a), during application of 20mM heplanol (b,c) and after wash out (d). In Bpercenlages
of p-,b- and n-WCi are shown. N =total number of EJCs. S is Ihe stimulation arcifact. p-. b- and n- EJC as explained in the text. The nerve a d c m potential (n.a.p.)
preceding the ETC in each case is not clearly visible in the records in A bemuse of noise and aliasing in printing. However. in C they are clearly visible because
ofimproved tigndtonoirc ratio(35 records averaged). Stimulation at5V. 1OOps.O.7Hz.Notethesu~ressionofthep-EICandtheahancementofthepercmlage
of oca" (B)and amplitude (C) of the n-EJC in the presence of heptanol.
+l...+
A
Fig.2 Single unit nerve aaion potential (nap.) before (A) and in h e presence
ACKNOWLEDGEMENTS
Financial support from the Department of Science and
Technology, India, under project no. SPISOINO6193 is gratefully acknowledged.
(B) of 20 mM hepcPnol (2-3min. application). Each trace is the average of
10 records.
underneath thc electrode. This will produce a p-EJC, which by
superposition will tend to attenuate an n-EJC arising simultaneously. If heptanol uncouples smooth muscle cells by blocking
intercellularelectricaljunctions [4], the positivity may no longer
be sensed by the recording electrode. Its amplitude in both the
p- and b-EJC will therefore be suppressed, resulting concomitantly in enhancement of the n-EJC, as observed in these
experiments.
The pEJC could also be suppressed by heptanol-induced
block of neurotransmitter release or action only outside the
recording electrode but not inside, if heptanol were unable to
gain access to the electrode interior due to the electrode-tissue
seal. However, as shown by the recordings of the nerve action
potential (Fig. 2). heptanol does not Seem to affect any of the
processes leading up to transmitter release. Furlhermore, evidence from other studiesindicates that heplanol does not elfect
neurotransmitter release or its post synaptic action in this tissue
[8],and that it specifically uncouples smooth muscle cells,
leaving other properties unaffected [4]. It is therefore likely that
the effects of hepmol on the phasicity of EJCs are related to its
property of uncoupling syncytial cells.
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